The present invention relates to a laser microscopy technique which produces molecular excitation in a target material by simultaneous absorption of three or more photons. The invention is an improvement over the two-photon laser microscopy technique disclosed in U.S. Pat. No. 5,034,613 to Denk et al. (hereinafter, the ""613 patent), and this patent is hereby incorporated by reference.
The ""613 patent discloses a laser scanning microscope which produces molecular excitation in a target material by simultaneous absorption of two photons to provide intrinsic three-dimensional resolution. Fluorophores having single photon absorption in the short (ultraviolet or visible) wavelength range are excited by a stream of strongly focused subpicosecond pulses of laser light of relatively long (red or infrared) wavelength range. The fluorophores absorb at about one half the laser wavelength to produce fluorescent images of living cells and other microscopic objects. The fluorescent emission from the fluorophores increases quadratically with the excitation intensity so that by focusing the laser light, fluorescence and photobleaching are confined to the vicinity of the focal plane. This feature provides depth of field resolution comparable to that produced by confocal laser scanning microscopes, and in addition reduces photobleaching. Scanning of the laser beam, by a laser scanning microscope, allows construction of images by collecting two-photon excited fluorescence from each point in the scanned object while still satisfying the requirement for very high excitation intensity obtained by focusing the laser beam and by pulse time compressing the beam. The focused pulses also provide three-dimensional spatially resolved photochemistry which is particularly useful in photolytic release of caged effector molecules.
A drawback to the two-photon laser microscopy technique disclosed in the ""613 patent is that its applications are limited by the available laser technology. In particular, the two-photon technique requires use of a laser at specific wavelengths, depending upon the application, so that the sum of energy levels of the two photons provides the specific energy level needed to generate the desired fluorescent emission. Unfortunately, some laser microscopy applications would require use of a laser having a wavelength which is not technologically feasible at the present time. For example, excitation of chromophores that have very short wavelength absorption, such as amino acids and nucleic acids, would require a laser having a 540 nm wavelength using the two-photon technique, and such a laser does not exist at the present time.
The present invention provides a solution to the aforementioned problem through the application of three or more photon excitation to laser scanning fluorescence microscopy and to spatially resolved photo-chemical processing, such as caged reagent activation for micropharmacology and polymer cross linking for 3-d optical information storage.
Because three-photon induced fluorescence obeys a cubic dependence on excitation intensity and four photon excitation obeys a quartic dependence, both provide intrinsic three-dimensional resolution in laser scanning microscopy. Although such 3-d resolution has already been achieved by the nonlinear microscopy technique based on two-photon excitation disclosed in the ""613 patent, three-photon excitation provides a unique opportunity to excite molecules normally excitable in the UV range (230-350 nm) with near IR light (700-1100 nm). Interesting biomolecules, such as the amino-acids tryptophan and tyrosine, the neurotransmitter serotonin and nucleic acids, have one-photon absorption peaks at approximately 260-280 nm, and fluorescence can be excited in these biomolecules by three and four photon excitation. The advantages of using long wavelength, near IR light are possibly less photodamage to living cells and conveniently available solid state femtosecond laser sources for deep UV absorbers. In practice, the configuration of three-photon laser scanning microscopy can be identical to the existing two-photon systems. However, because three-photon and two-photon absorption spectra are in general quite different, the combination of two- and three-photon excited fluorescence microscopy extends the useful range of the laser systems currently employed in two-photon microscopy.
A particularly advantageous application of three or more photon excitation is the replacement of excimer lasers for certain applications which require absorption of wavelengths around 200 nm. Three and four photon excitation by lasers generating much longer wavelengths (say 550-900 nm) should provide similar energy absorption and provide 3-d spatial resolution as well. Because excimer lasers are extremely expensive and user unfriendly, several photon excitation could be highly desirable.
The practicality of the proposed three-photon microscopy depends crucially on the three-photon fluorescence excitation cross-sections of various fluorophores and biomolecules. However, very few three-photon absorption cross-sections have been reported. A simple calculation based on perturbation theory shows that three-photon excitation would typically need  less than 10 times the peak intensity currently used with two-photon excitation technique to achieve a comparable level of excitation. This required intensity level can be easily accessed by femtosecond laser sources, such as the modelocked Ti:sapphire laser. Three-photon induced fluorescence of tryptophan and serotonin has been observed at excitation wavelengths between approximately 800 and 900 nm using a modelocked Ti:sapphire laser. The measured fluorescence obeys an expected cubic law dependence on excitation intensity. Measurements of fluorescence power of the calcium indicator dye Fura II at an excitation wavelength (approximately 911 nm) well below the expected three-photon excitation optimum, showed that satisfactory fluorescence images should be obtainable at only xcx9c5 times the laser power required for two photon excitation of Fura II at its optimum excitation wavelength (approximately 730 nm). The estimated three-photon fluorescence excitation cross-section from these preliminary results shows that three-photon laser scanning microscopy can be done with a reasonable level of excitation power. How widely applicable this approach will be remains to be determined. Four-photon excitation may be limited by the onset of strong one-photon absorption by water above about 1000 nm.
Studies of molecular excitation of fluorescence by three or more photon processes are rare because the excitation cross sections have been expected to be quite small. Thus, useful rates of excitation usually require very high instantaneous illumination intensities. A simple extrapolation of multiphoton cross sections is suggested by the pattern of matrix elements products in the perturbation theory solutions of the quantum mechanics of the dipole transition probability for molecular excitation by a radiation field. Basically, the multiphoton processes require three or more photons to interact with the molecule (within the cross sectional area of a molecule, Axcx9c10xe2x88x926 cm2) and simultaneously (within a time interval determined by the life times of intermediate states, xcex4xcfx84xcx9c10xe2x88x9216s). This short coincidence time is limited by the large energy uncertainties introduced by the perturbation theory energy denominators.
Fluorescence excitation by several photons does not significantly increase laser microscopy resolution because the longer excitation wavelength (for a given fluorophore) decreases resolution by about as much as it is increased by raising the one-photon point spread function to the power n for several-photon processes. Were it not for the wavelength factors, the increase in resolution of three photon excitation would be essentially the same as that incurred by adding an ideal confocal spatial filter to two photon microscopy.
Recent reports of unexpectedly large three-photon cross sections have been found in the course of research directed toward enhancing optical limiting absorption (which is intended to provide variable shades for protection of human vision from excessively brilliant light flashes). Recently, a three photon absorption cross section of about 10xe2x88x9275 cm6s2 has been reported for absorption and fluorescence of 2,5-benzothiazo 3,4-didecyloxy thiophene in tetrahydrofuran. Another recent experiment shows three photon excitation of fluorescence from a conjugated organic polymer. This process, however, appears to involve two excitation states unlike most fluorescence excitation. Although the conditions of these experiments are hardly suitable for laser scanning fluorescence microscopy or for most microphotochemistry, the large cross sections are promising. Note, however, that such large cross-sections are not essential for three or more photon microscopy.
The excitation wavelength dependence of the rate of photodamage to living cells during fluorescence microscopy and photo-micropharmacology is largely unknown and may vary greatly for different applications. Empirical studies have shown that two-photon excitation elicits far less damage than one-photon excitation for comparable fluorescence image acquisition. It is not clear whether further improvement can be made by stepping up to three- or four-photons for excitation. With the aid of such knowledge and the knowledge of the nonlinear absorption spectra, it is conceivable that the optimum excitation mode can be determined and utilized for each individual system in the future. A particularly appealing possibility is the use of one laser wavelength to induce photochemistry by three photon excitation and concurrently two-photon excitation of an accompanying fluorescence signal. Three-photon excitation seems quite likely to become a useful enhancement of the existing two-photon excitation technique but seems unlikely to replace it.
Alternatively, for microscopic photochemical activation, photoablation and optical surgery, the photo excitation can be advantageously accomplished by multi-photon excitation of intrinsic chromophores or even added chromophores that have very short wavelength absorption such as amino acids and nucleic acids. Multi-photon excitation allows the selection of more available lasers providing subpicosecond pulses at long wavelengths and long wavelength light transmission to the microscopic focal volume where photo excitation is desired.